无标签定量蛋白经济学工作流程，用于发现驱动的宿主-病原体交互
Summary
在这里，我们提出了一个协议，以分析宿主和病原体之间的相互作用，在感染基于质谱学的蛋白组学。此协议使用无标签量化来测量宿主（例如巨噬细胞）和病原体（例如 新福尔曼隐球菌）的蛋白质丰度变化。
Abstract
基于质谱学（MS）的定量蛋白质组学的技术成就为分析生物体在不同条件下的全球蛋白质组开辟了许多未被发现的途径。这种强大的策略应用于微生物病原体与所需宿主的相互作用，全面地描绘了两种感染视角。在此，工作流描述了 密码球菌新福尔曼的 感染物的无标签量化（LFQ），这是一种真菌诱发性细胞内病原体，是致命疾病隐科病的致病剂，存在不朽的巨噬细胞。该协议在单个实验中详细说明了病原体和哺乳动物细胞的适当蛋白质制备技术，从而为液相色谱 （LC） -MS/MS 分析提交了适当的肽。LFQ 的高通量通用性允许广泛的蛋白质识别和量化动态范围，以及可转移到任何宿主病原体感染设置，保持极端敏感性。该方法进行了优化，以编目感染模仿条件下病原体的广泛、公正的蛋白质丰度配置文件。具体来说，这里演示的方法提供了关于 C.新福尔曼病 原体的基本信息，例如毒性所需的蛋白质生产，并识别出对微生物入侵作出反应的关键宿主蛋白。
Introduction
侵入性真菌感染的流行率正在大大增加，与不可接受的高死亡率有关，最常见的报告在有免疫缺陷倾向1的个人。隐球菌是一种臭名昭著的机会性真菌病原体，能够在宿主巨噬细胞内细胞内细胞内存活。抗真菌干预不足导致真菌传播和危及生命的隐球菌脑膜炎和脑膜炎2，3。全球免疫功能化地位的提高要求同时增加抗真菌剂的使用，其中许多真菌物种，包括C.新福曼，已经逐渐进化到4，5，6的抗药性。因此，必须实施有力和有效的技术，以回答有关宿主防御反应和微生物发病机制的重要生物问题。
质谱学技术进步的新时代，包括强大的计算和生物信息管道的产生，为大规模分析宿主病原体研究提供了基础。传统的发病机制驱动的蛋白质组分析通常从宿主或病原体的角度描述感染的观点，包括综合方法，如蛋白质相关性分析，亲和色谱结合蛋白质组学，和相互作用经济学9。对宿主系统中危险病原体毒性的调查具有巨大的临床意义：然而，在单个实验中应用双重透视分析以前被认为是无法实现的。例如，病原体对感染的看法往往被高度丰富的宿主蛋白所淹没，导致检测低富真菌蛋白7的敏感性降低。此外，高样本复杂性邀请许多目标在单个实验系统中进行调查，并证明很难阐明特定病原体蛋白的行动机制。
自下而上的蛋白组学是一种流行的MS技术，它使可管理的样品制备，其中肽产生序列特异性酶消化，然后液体色谱分离，识别和量化MS10，11。在这里，我们演示了一种基于数据的获取策略的方法，旨在实现基于感染的蛋白质组或”感染体”的公正覆盖。具体来说，无标签量化（LFQ）可以摆脱对化学或代谢标签的依赖，从而对多个蛋白质组的蛋白质水平变化进行可靠和准确的识别，从而减少样品处理和处理步骤12、13。这种普遍应用在细胞内的某一时刻询问产生的蛋白质，而该细胞独立于任何预期的蛋白质生产：因此，可能会发现对感染至关重要的新见解。
此处描述的工作流程进行了优化，以探索 C.新福尔曼在与 宿主免疫细胞的感染模仿条件下的蛋白质水平变化（图1）。这种方法不是依靠细胞类型的分离和分离，而是将宿主和病原体蛋白质组分离在一起，并利用生物信息分离利用两个生物特定的数据库来区分特定物种的蛋白质生产。这种方法为无限数量的样品处理提供了优势，无需同位素标签研究或分数所需的额外成本高昂的制备步骤。此外，此工作流程支持可转移到各种真菌和细菌病原体的优化蛋白质提取方案，这些病原体能够瞄准和感染宿主免疫细胞。总体而言，该协议概述了完成高分辨率MS的公正蛋白质提取和样品处理的步骤，然后是数据和统计分析，能够提供大量对感染具有重要意义的真菌蛋白知识，并结合对宿主防御反应的全面分析。
Protocol
圭尔夫大学动物利用议定书4193批准的以下议定书使用了源自BALB/c小鼠的永生巨噬细胞系。值得注意的是，其他小鼠菌株或其他永生细胞来源可以应用于概述的协议，并有足够的测试，以优化详细的参数。以下协议将导航从大噬细胞的冷冻小瓶开始的步骤。细胞储存在10S（胎儿牛血清），1%L-谷氨酰胺和5%笔/链球菌（青霉素-链霉素）混合物DMEM（杜尔贝科的改性鹰介质）和20%DMSO（二甲基硫氧化物?…
Representative Results
上述协议允许在单个实验中识别和量化来自真菌病原体C.新福尔曼和宿主巨噬细胞的蛋白质。在共同培养之后，细胞被收集和处理在一起，并根据每个物种特有的肽谱进行生物信息分离。这是定义感染期间宿主-病原体关系相互作用的有力方法。从实验中识别出的蛋白质数量取决于启动材料、样品准备、梯度长度、MS 仪器和生物信息工作流程。使用此处描述的协议，我们通常从实验中识别出?…
Discussion
协议中的关键步骤包括制备巨噬细胞和收集用于蛋白质加工的共同培养样本，尽量减少对细胞的干扰。重要的是要执行步骤，清洗，接种，并删除粘附的巨噬细胞轻轻地和仔细，以防止不必要的裂解细胞之前收集。建立正确的实验MOI也至关重要，因为接种过高的MOI会导致快速的巨噬细胞死亡和MS的收集和处理样本的困难。 相反，低MOI数量将导致噬菌体真菌细胞减少，生物系统中真菌蛋白的检测有?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢生物信息学解决方案公司的乔纳森·克里格博士为具有代表性的实验操作质谱仪，以及盖德斯-麦卡利斯特小组的成员在实验设置和手稿反馈方面给予的帮助。作者承认部分资金支持 来自班廷研究基金会-贾里斯洛夫斯基奖学金发现奖，新前沿研究基金-探索（NFRFE-2019-00425），加拿大创新基金会（JELF 38798）为J.G.M，以及NSERC加拿大研究生奖学金-硕士和安大略研究生奖学金的B.B，和女王伊丽莎白二世研究生奖学金为A.S.。
Materials
| 100 mM Tris-HCl, pH 8.5 | Fisher Scientific | BP152-1 | Maintain at 4°C |
| 60 x 15 mm Dish, Nunclon Delta | ThermoFisher Scientific | 174888 | |
| 6-well cell culture plate | ThermoFisher Scientific | 140675 | |
| Acetonitrile, MS grade | Pierce | TS-51101 | |
| Acetic Acid | Sigma Aldrich | 1099510001 | |
| Acetone | Sigma Aldrich | 34850-1L | |
| Ammonium bicarbonate (ABC) | ThermoFisher Scientific | A643-500 | Prepare a stock 50 mM ABC solution, stable at room temperature for up to one month. |
| Bel-Art™ HiFlow Vacuum Aspirator Collection System | Fisher Scientific | 13-717-300 | Not essential, serological pipettes can be used to remove media. |
| C18 resin | 3M Empore | 3M2215 | |
| Cell Scrapers | VWR | 10062-906 | Not essential, other methods to release macrophage cells can be used. |
| Centrifugal vaccuum concentrator | Eppendorf | 07-748-15 | |
| Complete Filtration Unit | VWR | 10040-436 | |
| Conical falcon tubes (15 mL) | Fisher Scientific | 05-539-12 | |
| Countess II Automated Cell Counter | ThermoFisher Scientific | AMQAX1000 | Not essential, haemocytometer can be used as an alternative. |
| CytoTox 96 Non-Radioactive Cytotoxicity Assay | Promega | G1780 | |
| Dithiothreitol (DTT) | ThermoFisher Scientific | R0861 | Prepare bulk stock solution of 1 M DTT, flash frozen and stored at -20 °C until use. Discard after each use (do not freeze-thaw repeatedly). |
| DMEM, high glucose, GlutaMAX Supplement | ThermoFisher Scientific | 10566016 | |
| Fetal Bovine Serum (FBS) | ThermoFisher Scientific | 12483020 | Heat inactivate by incubating at 60°C for 30 minutes. Prepare 50 ml aliquots and flash freeze. Thaw prior to media preparation |
| Haemocytometer | VWR | 15170-208 | |
| HEPES | Sigma Aldrich | H3375 | Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20°C until use. Discard after each use (do not freeze-thaw repeatedly). |
| High-performance liquid chromatography system | ThermoFisher Scientific | LC140 | Gradient length is based on sample complexity, recommended 120 min gradient for infectome samples. |
| High-resolution mass spectrometer | ThermoFisher Scientific | 726042 | |
| Iodoacetamide (IAA) | Sigma Aldrich | I6125 | Prepare 0.55 M bulk stock solution, flash frozen, store at -20°C until use. Discard after each use (do not freeze-thaw repeatedly). |
| L-glutamine | ThermoFisher Scientific | 25030081 | Can be aliquot and frozen for storage. Thaw prior to media preparation. |
| LoBind Microcentrifuge tubes | Eppendorf | 13-698-794 | |
| MaxQuant | https://maxquant.org/ | MaxQuant is a public platform that offers tutorials, such as the MaxQuant Summer School, outlining the computational analysis steps of large MS data sets | |
| Microcentrifuge | Eppendorf | 13864457 | |
| Penicillin : Streptomycin 10k/10k | VWR | CA12001-692 | Can be aliquot and frozen for storage. Thaw prior to media preparation. |
| Peptide separation columns | ThermoFisher Scientific | ES803 | |
| Perseus Software | http://maxquant.net/perseus/ | ||
| Phosphate Buffered Saline | VWR | CA12001-676 | Puchase not required. PBS can also be prepared but sterile filteration must be performed before use. |
| Pierce BCA Protein Assay | ThermoFisher Scientific | 23225 | |
| Pipette, Disposable Serological (10 mL) | Fisher Scientific | 13-678-11E | |
| Pipette, Disposable Serological (25 mL) Basix | Fisher Scientific | 14955235 | |
| Probe sonciator | ThermoFisher Scientific | 100-132-894 | |
| Protease inhibitor cocktail tablet | Roche | 4693159001 | |
| Sodium dodecyl sulfate | ThermoFisher Scientific | 28364 | 20% (w/v) |
| Spectrophotometer (Nanodrop) | ThermoFisher Scientific | ND-2000 | |
| STAGE tipping centrifuge | Sonation | STC-V2 | |
| Thermal Shaker | VWR | NO89232-908 | |
| Trifluoroacetic acid | ThermoFisher Scientific | 85183 | |
| Trypsin/Lys-C protease mix, MS grade | Pierce | A40007 | Maintain at -20 °C. |
| Ultrasonic bath | Bransonic | A89375-450 | Stored in cold room (4C) |
| Urea | Sigma Aldrich | U1250-1KG | Prepare 40 mM HEPES/8 M Urea in bulk stock solution, flash frozen, store at -20 °C until use. Discard after each use (do not freeze-thaw repeatedly). |
| Yeast-extract peptone dextrose broth | BD Difco | BM20 |
References
- Bongomin, F., Gago, S., Oladele, R., Denning, D. Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. Journal of Fungi. , (2017).
- Tugume, L., et al. HIV-Associated cryptococcal meningitis occurring at relatively higher CD4 counts. Journal of Infectious Diseases. 219 (6), 877-883 (2019).
- Rajasingham, R., et al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. The Lancet Infectious Diseases. , (2017).
- Perfect, J. R. The antifungal pipeline: A reality check. Nature Reviews Drug Discovery. , (2017).
- Bermas, A., Geddes-McAlister, J. Combatting the evolution of anti-fungal resistance in Cryptococcus neoformans. Molecular Microbiology. , 1-14 (2020).
- Geddes-McAlister, J., Shapiro, R. S. New pathogens, new tricks: Emerging, drug-resistant fungal pathogens and future prospects for antifungal therapeutics. Annals of the New York Academy of Sciences. , (2018).
- Ball, B., Bermas, A., Carruthers-Lay, D., Geddes-McAlister, J. Mass Spectrometry-Based Proteomics of Fungal Pathogenesis, Host-Fungal Interactions, and Antifungal Development. Journal of Fungi. , (2019).
- Sukumaran, A., et al. Decoding communication patterns of the innate immune system by quantitative proteomics. J Leukocyte Biol. , (2019).
- Salas, D., Stacey, R. G., Akinlaja, M., Foster, L. J. Next-generation interactomics: Considerations for the use of co-elution to measure protein interaction networks. Molecular and Cellular Proteomics. , (2020).
- Aebersold, R., Mann, M. Mass-spectrometric exploration of proteome structure and function. Nature. 537 (7620), 347-355 (2016).
- Mann, M., Kulak, N. A., Nagaraj, N., Cox, J. The Coming Age of Complete, Accurate, and Ubiquitous Proteomes. Molecular Cell. 49 (4), 583-590 (2013).
- Cox, J., et al. Accurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ. Molecular & Cellular Proteomics. 13 (9), 2513-2526 (2014).
- Ankney, J. A., Muneer, A., Chen, X. Relative and Absolute Quantitation in Mass Spectrometry-Based Proteomics. Annual Review of Analytical Chemistry. , (2018).
- Cox, J., Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnology. 26 (12), 1367-1372 (2008).
- Tyanova, S., et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nature Methods. 13 (9), 731-740 (2016).
- Cox, J., et al. Andromeda: A peptide search engine integrated into the MaxQuant environment. Journal of Proteome Research. , (2011).
- Zhong, Z., Pirofski, L. A. Opsonization of Cryptococcus neoformans by human anticryptococcal glucuronoxylomannan antibodies. Infection and Immunity. , (1996).
- Nicola, A. M., et al. Macrophage autophagy in immunity to Cryptococcus neoformans and Candida albicans. Infection and Immunity. , (2012).
- Ball, B., Geddes-McAlister, J. Quantitative Proteomic Profiling of Cryptococcus neoformans. Current Protocols in Microbiology. , (2019).
- Geddes-McAlister, J., Gadjeva, M. Mass spectromerty-based quantitative proteomics of murine-derived polymorphonuclear neutrophils. Current Protocols in Immunology. , (2019).
- Geddes, J. M. H., et al. Secretome profiling of Cryptococcus neoformans reveals regulation of a subset of virulence-associated proteins and potential biomarkers by protein kinase A. BMC Microbiology. , (2015).
- Geddes, J. M. H., et al. Analysis of the protein kinase a-regulated proteome of Cryptococcus neoformans identifies a role for the ubiquitin-proteasome pathway in capsule formation. mBio. 7 (1), 1-15 (2016).
- Al Shweiki, M. H. D. R., et al. Assessment of Label-Free Quantification in Discovery Proteomics and Impact of Technological Factors and Natural Variability of Protein Abundance. Journal of Proteome Research. , (2017).
- Ong, S. E. Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Molecular & Cellular Proteomics. 1 (5), 376-386 (2002).
- Thompson, A., et al. Tandem mass tags: A novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Analytical Chemistry. 75 (8), 1895-1904 (2003).
- Borner, G. H. H. Organellar Maps Through Proteomic Profiling – A Conceptual Guide. Molecular & Cellular Proteomics. , (2020).
- Gingras, A. C., Abe, K. T., Raught, B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Current Opinion in Chemical Biology. , (2019).
- Kugadas, A., et al. Frontline Science: Employing enzymatic treatment options for management of ocular biofilm-based infections. Journal of Leukocyte Biology. , (2019).
- Yeung, J., Gadjeva, M., Geddes-McAlister, J. Label-Free Quantitative Proteomics Distinguishes General and Site-Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface. Proteomics. , (2020).
- Yeung, J., Lamb, J., Krieger, J. R., Gadjeva, M., Geddes-McAlister, J. Quantitative Proteomic Profiling ofMurine Ocular Tissue and theExtracellular Environment. Current Protocols in Mouse Biology. 10 (83), (2020).
Cite This Article
Ball, B., Sukumaran, A., Geddes-McAlister, J. Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions. J. Vis. Exp. (164), e61881, doi:10.3791/61881 (2020).